(I) ATP is a source of intracellular energy as well as a substance deeply involved in the life process. On the other hand, firefly luciferase catalyzes the reaction of forming oxyluciferin, CO2, AMP, and pyrophosphoric acid through D-luciferin as a luminescent substrate in the presence of ATP, O2, and Mg2+, thereby producing luminescence. Further, the luminescent reaction of luciferase depends on the amount of ATP.
Therefore, an assay to quantify ATP using the luminescent reaction of luciferase has been used since a long time ago. In fields, such as biotechnology, clinical laboratory test, and food hygiene, methods for measuring the amount of intracellular ATP using luciferase have been developed.
For example, the amount of intracellular ATP is usually measured by the following steps (1-1) to (1-3):
(1-1) dissolving cells or bacteria to extract ATP;
(1-2) adding the extracts to a reaction solution containing luciferin and luciferase; and
(1-3) quantifying the amount of intracellular ATP by measuring the luminescence from the reaction solution to which the extracts are added.
The amount of ATP in the cells which are not homogenized is usually measured by the following steps (2-1) to (2-3):
(2-1) introducing a luciferase gene into cells to obtain expression thereof;
(2-2) adding luciferin to a culture solution containing cells; and
(2-3) quantifying intracellular ATP by measuring the luminescence from the culture solution to which luciferin is added.
The serial measurement of the amount of ATP at a predetermined site (specifically, mitochondria) in living cells is performed by the following steps (3-1) and (3-2) (Non-patent document 1):
(3-1) fusing a mitochondrial targeting signal gene to a luciferase gene and introducing the fused gene into cells; and
(3-2) sequentially measuring changes of the amount of ATP in mitochondria in the cells by measuring the luminescence from the cells on the presupposition that luciferase is localized in mitochondria in the cells.
Since the intensity of luminescence emitted from cells is very weak, photon counting is performed by using a CCD camera equipped with an image intensifier to recognize one cell. Cells other than the cells whose amount of luminescence is measured are used to determine whether luciferase is localized in mitochondria in cells or not. Specifically, the different cells are immobilized and reacted with anti-luciferase antibodies, and then the cells are observed by a fluorescent antibody method in order to confirm their localization. As a result, it is suggested that the amount of luminescence from the measured cells corresponds to the amount of luminescence from mitochondria.
(II) The cell proliferation is one of the essential and important characteristics for organisms in the vital life processes. The cell cycle includes multiple consecutive reactions consisting of the growth of cells, the DNA duplication, the distribution of chromosomes, the cell division, and the like. Therefore, it is just conceivable that the expression of various genes varies depending on each stage of the cell cycle. Further, it is considered that abnormality or disruption of the cell cycle is involved in numerous chronic diseases and oncogenesis (refer to Patent document 1). In addition, Patent document 1 discloses a technique relative to a method of measuring the activity of a cell-cycle regulator and a method of diagnosing cancer using thereof.
Incidentally, when the luciferase gene is introduced into cells as a reporter gene and the strength of expression of the luciferase gene is examined using the luciferase activity as an indicator, the effect of a target DNA fragment on the transcription of the luciferase gene can be examined by linking the target DNA fragment to upstream or downstream of the luciferase gene. Further, a gene such as a transcription factor, considered to affect the transcription of luciferase gene, is linked to an expression vector and coexpressed with the luciferase gene, thereby enabling to examine the effect of a gene product of the gene on the expression of the luciferase gene. In this regard, examples of the method of introducing a reporter gene such as a luciferase gene into cells include a calcium phosphate method, a Lipofectin method, and an electroporation method. Each method is used separately depending on the purpose or the difference in the type of cell.
Further, the activity of the luciferase which is introduced into cells and is expressed is measured (monitored) by the steps of reacting a cell lysate in which the cells are dissolved with a substrate solution containing luciferin, ATP, magnesium, or the like, and then quantifying the amount of luminescence from the cell lysate reacted with the substrate solution using a luminometer with a photomultiplier tube. That is, the luminescence is measured after dissolving the cells. Thus, the amount of expression of the luciferase genes at a certain point in time can be measured as an average value of the whole cells.
In order to catch the amount of expression of luciferase genes with time, it is necessary to measure the luminescence from living cells sequentially. The serial measurement of the luminescence from living cells is performed by the steps of adding a luminometer function to an incubator for culturing cells, and then quantifying the amount of luminescence from whole cell populations while culturing at regular time intervals using a luminometer. This allows for measuring the expression rhythm with a regular cycle, and the like. Thus, it is possible to catch changes over time of the amount of expression of luciferase genes in whole cells.
However, in the conventional reporter assay as described above, multiple cells at different stages of the cell cycle are mixed, so that the cells at various stages have been handled as a group of data. Therefore, the operation of synchronized culture is performed to match the stages of the cell cycle when the gene in connection with the cell cycle is analyzed.
(III) Conventionally, a microscope apparatus that can observe a specimen by switching imaging magnification of a specimen image from a high magnification mode to a low magnification mode, has commonly been used. With reference to such a microscope apparatus, there has been recently proposed a microscope apparatus in which a visual field of observation at low magnification is not limited by an objective lens with high magnification and the overall image of a specimen can be grasped in a wider visual field (for example, refer to Patent document 2). In the microscope apparatus disclosed in Patent document 2, when a specimen is observed at low magnification, the specimen image is formed using an imaging lens for low magnification, in which the focal depth is deeper than the conventional one, namely, NA (Numerical Aperture) on the side of the specimen is smaller than the conventional one without the objective lens with high magnification.    Patent document 1: Japanese Patent Application Laid-Open (JP-A) No. 2002-335997    Patent document 2: JP-A No. 10-339845    Non-patent document 1: H. J. Kennedy, A. E. Pouli, E. K. Ainscow, L. S. Jouaville, R. Rizzuto, G. A. Rutter, “Glucose generates sub-plasma membrane ATP microdomains in single islet β-cells.”, Journal of Biological Chemistry, vol. 274, pp. 13281-13291, 1999